Throughout this application various publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Serotonin, 5-hydroxytryptamine (5HT), is a biogenic amine that functions as a neurotransmitter (12), a hormone (13), and a mitogen (14). Serotonin modulates many forms of synaptic transmission and is believed to exert a number of effects on the growth of neurons in early development. In the spinal cord, serotonin is involved in the inhibitory control of sensory input and in the facilitation of motor output (15, 16). In the cortex, transmission at serotonergic synapses contributes to affective and perceptual states, and these synapses represent a major site of action of psychotropic drugs such as LSD (17). Serotonergic neurons project to diffuse regions of the brain and exert their physiological effects by binding to cell surface receptors. To date, six serotonin receptor subtypes (5HT1a-1d, 2 and 3) (previously designated 5HT-1A-1D, 2 and 3) have been defined on the basis of their pharmacological properties (18).
Individual receptor subtypes reveal characteristic differences in their abilities to bind a number of ligands, but the structural basis for the distinct ligand-binding properties is not known. Physiologists and pharmacologists have attempted to specify particular biological functions or anatomical locations for some receptor subtypes, but this has met with limited success.
Similarly, the biochemical mechanisms by which these receptors transduce signals across the cell surface have been difficult to ascertain without having well-defined cell populations which express exclusively one receptor sub-type. Serotonin receptor subtypes couple to different intracellular second messenger signaling systems, including the regulation of adenylate cyclase activity (5HT1a and 5HT1b) (19, 20, 21) and phospholipase C activities (5HT1c and 5HT2) (22, 23). The activation of these second messenger pathways by serotonin modulates the excitable properties of both central and peripheral neurons (24, 25, 26, 27). Serotonin receptors are also thought to be linked to the direct modification of ion channel states, and are implicated in mechanisms associated with pain, migraine headaches, and motor control. Moreover, drugs which bind to the serotonin receptor may be useful in treating depression. One difficulty which this involves is the prior difficulty in examining a specific interaction of a drug with the serotonin 5HT1c receptor alone.
The methods provided by this invention provide a simple and qualitative assay to assess this interaction and eliminate the lack of specificity associated with the prior art use of tissue preparations as a semi-defined source of serotonin receptors. The expression of functional receptors in Xenopus oocytes has provided a sensitive assay for detection of mRNA encoding serotonin receptors, in particular the 5HT1c receptor, that couples via inositol phospholipid signaling systems (5, 6, 28). This invention differs from the closest prior art in that NIH 3T3-SR cells can be obtained in any quantity desired, and provides the investigator with a source of receptors which is consistent in its molecular characteristics. Furthermore, this cell line also provides the investigator with a cellular environment in which ligand-receptor interactions can be measured using a simple spectrofluorimetric assay.
Although it has recently been reported that a cDNA fragment encoding the serotonin 5HT1c receptor has been cloned, this fragment does not encode and is not useful in producing functional serotonin 5HT1c receptors. The cDNA clone encodes only the carboxyl-terminal portion of the 5HT1c receptor and was isolated by hybrid-depletion of choroid plexus mRNA coupled with oocyte expression (29). Applicants, in the present invention, have combined cloning in RNA expression vectors with an electrophysiological assay in oocytes to isolate and characterize the expression of a functional cDNA clone encoding the entire 5HT1c receptor.